Nitrogen is a critical limiting nutrient for plants. Nitrogen fertilizer is a significant contributor to the yield increases obtained in the last several decades. However, these yield benefits have monetary and environmental costs, and nitrogen-based fertilizer represents a significant fraction of a farmer's input costs. Furthermore, crops only use a fraction of applied nitrogen. For example, it has been estimated that 50-70% of the nitrogen provided to the soil is lost (Masclaux-Daubresse et al., 2010, Ann. Bot. 105: 1141-1157; Hodge et al., 2000, Trends Plant Sci. 5: 304-308). Maize production in the US is reported to have a nitrogen fertilizer recovery efficiency of 37% (Cassman et al., 2002, Ambio 31: 132-140), and increased fertilizer application rates are subject to diminishing returns. A hectare of corn, for example, retains 39% of the first 100 kilograms of nitrogen applied as fertilizer, but only 13% of the second 100 kilograms of nitrogen applied (Socolow, 1999, Proc. Natl. Acad. Sci. USA 96: 6001-6008). As a consequence, nitrogen fertilizer that is not taken up by plants is generally lost as runoff or converted to nitrogen gases by microbial action, contributing to water and air pollution.
Thus, improving their efficiency of a crop plant's nitrogen use (i.e., its Nitrogen Use Efficiency, or N Use Efficiency, or NUE) would have the benefit of improving yield and agricultural sustainability while reducing negative environmental impact. NUE has been defined as increased grain yield per unit nitrogen available from the soil (Masclaux-Daubresse et al., 2010, supra), and thus it is judicious to identify means to increase the grain yield that may be obtained per unit nitrogen available from the soil.
Plants obtain nitrogen through the processes of uptake and assimilation (Buchanan et al., 2000, Biochemistry and Molecular Biology of Plants, American Society of Plant Physiologists, Rockville, Md.; Masclaux-Daubresse et al., 2010, supra). Uptake refers to the transport of nitrogen into the plant, and assimilation is the conversion of nitrate and ammonia to amino acids. Plants generally take up nitrogen from the soil in the form of nitrate or ammonium. Plants contain both low affinity and high affinity transport systems for these ions. In the case of nitrate, there is both a constitutive and an inducible high affinity transport system (Glass et al., 2002, J. Exp. Bot. 53: 855-864). Once nitrate crosses the plasma membrane, it is either metabolized in the cytoplasm of root cells or transported to the shoot via the xylem. For example, in wheat, up to 80% of the absorbed nitrate is reduced within the leaves (Ashley et al., 1975, Plant Physiol. 55: 1102-1106). Nitrate is reduced to ammonia through the action of nitrate reductase and nitrite reductase. Assimilation of ammonia takes place through the glutamine synthetase/glutamine-2-oxoglutarate aminotransferase (GS/GOGAT) pathway. Glutamine synthetase (GS) adds an amino group to glutamate to make glutamine, and GOGAT transfers the amino group to α-ketoglutarate to make a second molecule of glutamate. Photosynthesis provides the fixed carbon, energy, and reductant necessary for assimilation.
Plant nitrogen use efficiency could conceivably be increased by several mechanisms (Lawlor 2002, J. Exp. Bot. 53: 773-787). One mechanism could be increasing nitrogen uptake (which can be defined as the percentage of applied nitrogen taken up by plants (Maust and Williamson, 1994, J. Amer. Soc. Hort. Sci., 119: 195-201), through higher root surface area, deeper penetration into the soil, or more high affinity nitrate or ammonium transporters. A second mechanism could be increased assimilation, possibly by increased activity of assimilatory enzymes or removal of negative regulation. Nitrogen utilization or assimilation efficiency, NUtE, is the fraction of plant-acquired nitrogen to be converted to total plant biomass or grain yield; (Xu et al., 2012, Annu. Rev. Plant Biol. 63:153-182). A third mechanism could be increased capacity to store nitrogen when it is available. Nitrogen is stored in the form of nitrate in cell vacuoles, but stored nitrate supplies are exhausted in a matter of days (Glass et al., 2002, supra). Nitrogen is also stored in the form of amino acids and protein, and this storage is dependent upon sufficient carbon availability. Control of nitrogen losses is also possible. Nitrate and ammonia exit as well as enter root cells. Photorespiration is another source of ammonia loss. Ammonia released through photorespiration is recycled through the GS/GOGAT pathway, but this process may not be fully efficient. Overexpression of cytosolic glutamine synthetase in tobacco increased biomass produced, presumably through increased efficiency of ammonia recycling (Oliveira et al., 2002, Plant Physiol. 129: 1170-1180). The intrinsic nitrogen use efficiency (defined as biomass produced per unit N) could be changed by changing the plant's fundamental carbon/nitrogen ratio. Improving the NUE of crop plants has the potential to reduce fertilizer application rates, providing both cost savings and environmental benefits.
In spite of the apparent advantages of improved NUE, decades of research have not produced significant improvements in NUE in crops, and improved NUE is largely an unmet need in agriculture today.
The present description relates to methods and compositions for producing transgenic plants with modified traits, particularly traits that address agricultural and food needs by improving nitrogen use efficiency. In addition to reducing the demand for nitrogen application, it is expected that improving nitrogen use efficiency will improve yield and may provide significant value by allowing the plant to thrive in hostile environments, where, for example, low nutrient availability may limit yield or diminish or prevent growth of non-transgenic plants.
In this description, the expression levels of certain polynucleotide and polypeptide sequences identified herein may be manipulated to produce improved yield in commercially valuable plants and crops as well. Other aspects and embodiments of the description are described below and can be derived from the teachings of this disclosure as a whole.